Engineered CHO cells as a novel AAV production platform for gene therapy delivery

The Herpes simplex virus (HSV)-based platform for production of recombinant adeno-associated viral vectors (rAAVs) yields higher titers and increased percentage of full capsids when compared to the triple transient transfection (TTT) method. However, this platform currently faces two major challenges. The first challenge is the reliance on commercial media, sometimes supplemented with serum, leading to costly manufacturing and a high risk for introduction of adventitious agents. The second challenge is that the production of HSV-1 relies on adherent complementing Vero cells (V27), making it difficult to scale up. We engineered serum-free-adapted CHO cells expressing key HSV-1 entry receptors, HVEM and/or Nectin-1 to address the first challenge. Using high-throughput cloning methods, we successfully selected a HVEM receptor-expressing clone (CHO–HV–C1) that yields 1.62 × 109, 2.51 × 109, and 4.07 × 109 viral genome copies/mL with rAAV6.2-GFP, rAAV8-GFP, and rAAV9-GFP vectors respectively, within 24 h post rHSV-1 co-infection. Moreover, CHO–HV–C1-derived rAAVs had comparable in vitro transduction, infectivity, and biodistribution titers to those produced by TTT. The second challenge was addressed via engineering CHO–HV–C1 cells to express HSV-1 CP27. These cells successfully produced rHSV-1 vectors, but with significantly lower titers than V27 cells. Taken together, the CHO/HSV system provides a novel, scalable, reduced cost, serum-free AAV manufacturing platform.


rHSV-1 infection and rAAV9-GFP production in stable CHO pools
With the HVEM and Nectin-1 receptors stably expressed in CHO pools, it was critical to determine which of the eight constructs provided the best rHSV-1 infection and subsequent rAAV expression after coinfection with rHSV-1 vectors.Each of the stable pools generated above were infected with rHSV1-GFP at a multiplicity of infection (MOI) of 10 plaque forming units (PFU) per cell, with the wild-type CHO cells infected as a negative control.GFP expression data were collected from all the infected stable cell pools (n = 8) every 12 h post-infection (hpi) up to 60 h.All eight infected cell pools showed significant mean GFP expression starting at 12 hpi, when compared to the infected wild-type CHO cells (Fig. 2a).The highest level of GFP expression was observed at 12 hpi in the HVEM-only expressing pools (pools 1 and 2), with a p-value < 0.0001 when compared to the infected wild-type CHO cells.The next highest level of GFP expression came from Nectin-1-only expressing pools (pools 3 and 4), with a p-value < 0.016 when compared to the infected wild-type CHO cells (Fig. 2a).
To determine rAAV production from the stable pools, cell supernatant was collected a day after co-infection with rHSV-1 encoding AAV2 rep and AAV9 cap proteins, as well as rHSV-1 encoding GFP flanked by AAV2  ITRs, and rAAV9-GFP vector was titrated using qPCR.Pool 1 showed the highest rAAV9-GFP titer with an average of 1.58 × 10 8 vg/mL, compared to pools 2 through 8 that ranged between an average of 5.13 × 10 7 and 1.17 × 10 8 vg/mL, although this value was not significantly higher than the titers produced by the other pools.All stable pools produced more rAAV9-GFP than wild-type CHO cells (6.92 × 10 6 vg/mL, p < 0.0001 (Fig. 2b).These data indicate pool 1 outperformed all other pools for rHSV1-GFP infection, GFP expression, and rAAV-GFP production.Therefore, this pool was selected for a single-cell cloning.

Selection of top CHO clones for HSV-1 infection and AAV production
High and medium HVEM-expressing CHO cell subpopulations were identified and selected by fluorescence activated cell sorting (FACS) gating strategy and single cells from each of the gated populations (Supplementary Fig. S1)were deposited individually into a well of a 384-well plate.Deposition of single cell per well was verified by imaging using Cellavista 25 .After two weeks, 64 clones with a satisfactory outgrowth were selected for further evaluation.The selected clones were scaled up using 96-deep well plates and passaged using in-house medium supplemented with MSX.After three passages, 24 out of the initial 64 clones showed good HVEM expression by flow cytometry (Fig. 3a).These clones were expanded and further tested for rHSV1-GFP vector infection.The mean GFP expression, determined at 24 and 48 hpi, showed no significant difference among the tested clones (Fig. 3b).
Eight clones originated from CMV-HVEM-SV40 polyA (C1, C13, C15, C23, C24, C46, C62, and C64) showed the highest mean GFP expression post rHSV1-GFP infection.These eight clones were selected to further assess their capability to produce rAAV6.2-GFPvia co-infection at an MOI of 1:1 PFU/cell of rHSV-AAV2 rep/cap6.2and rHSV-GFP.Over the course of the co-infection, the viability of these eight clones decreased rapidly, compared to only a slight decrease (13%) of the infected wild-type CHO cells, when incubated at 37 °C (Fig. 4a).Likewise, the viable cell density (VCD) was also reduced for all co-infected clones compared to the minor VCD drop observed (by 0.3 × 10 6 cells/mL) for the infected wild-type CHO cells (Fig. 4b).It seems likely that the AAV rep protein exerts a cytotoxic effect on the infected engineered CHO cells, as previously reported for other cell lines such as HL-60 cells 26 .The cytotoxic effect is not observed in infected wild-type CHO host cells due to the degradation of rHSV-1 vectors after cellular entry.We found clone #1 (CHO-HV-C1) produced the highest rAAV6.2-GFPvector titer per mL of cell lysate (~ 6.76 × 10 8 vg/mL from 1 × 10 6 cells) at 24 hpi, compared to other clones that produced between 5.50 × 10 7 and 2.19 × 10 8 vg/mL.Although the rAAV6.2-GFPvector titer produced in the CHO-HV-C1 clone was not statistically different from titers produced by the other clones (p = 0.89), all clones had significantly higher titers than the co-infected wild-type CHO host cells, which produced 1.74 × 10 6 vg/mL at 24 hpi (p = 0.0015).It is also worth noting that for all eight co-infected clones, the rAAV6.2-GFPtiters decreased at 48 hpi.Interestingly, although CHO-HV-C1 is a high HVEM expressing clone and CHO-HV-C62 is a medium HVEM expressing clone, both produced the highest titers of rAAV6.2-GFPvector (Fig. 4c).Therefore, further investigation is necessary to determine if HVEM expression levels have any impact on rAAV production.
As CHO-HV-C1 was the most productive clone for rAAV6.2-GFPproduction, we wanted to further explore its capacity to produce rAAV8 and rAAV9 expressing the GFP transgene.Using the same optimal infection parameters described in the previous experiment, average titers of 1.62 × 10 9 and 2.51 × 10 9 vg/mL per 1 × 10 6 cells cell lysate for rAAV8-GFP and rAAV9-GFP vectors, respectively, were produced at 24 hpi.However, harvesting at 30 hpi resulted in decreased titers (8.36 and 9.55 × 10 8 vg/mL for rAAV8-GFP and rAAV9-GFP vectors, respectively; Fig. 4e).These data indicate that the infection parameters described above are suitable for a variety of serotypes, with optimal titer achieved at 24 hpi.Similar to rAAV6.2-GFPvector production, the co-infected CHO-HV-C1 cells for the production of rAAV8-GFP and rAAV9-GFP vectors showed a sharp decrease in viability at 24 hpi, indicating that the effect on cell viability is not serotype-specific.We hypothesized that a sharp decline in the cell viability after rHSV-1 vector co-infection may affect the final rAAV titers.Thus, anticipating an extension of culture duration we evaluated the effect of a temperature switch from 37 to 33 °C at the time of co-infection with rHSV-1 AAV6.2 and rHSV-1-GFP at MOI 4:1.At 24 hpi, the co-infected cultures with a temperature switch showed improved cell viability over cultures incubated at 37 °C (Supplementary Fig. S2a), together with a slight improvement in rAAV6.2-GFPtiters from cell lysate, as well as an increase in titer within the medium of approximately one log (Supplementary Fig. S2b).This finding indicates that post co-infection incubation temperature is an essential factor for cell viability and rAAV production in the CHO platform.

Analytical characterization of CHO-derived rAAVs
The PEG-Chloroform purification method has been reported as a fast, capsid-independent, and easy purification method for rAAV [27][28][29] .The entire bench-scale process of harvesting and purifying the rAAV vectors using PEG-Chloroform takes approximately one day (Supplementary Fig. S3).The final purified rAAV is stored at − 80 °C.In order to understand if VP expression ratios played a role in limiting rAAV production a higher MOI, purified rAAV were evaluated using Western blots.These purified rAAVs produced in CHO-HV-C1 clone using different MOIs of rHSV-1 showed comparable expression of VP1, VP2, and VP3 AAV capsid proteins (Fig. 5a).Moreover, examination of the purified rAAV6.2-GFPand rAAV9-GFP vectors produced in CHO cells using mini-TEM showed 91% and 79.5% full capsids, respectively (Fig. 5b,c), whereas the full capsid percentages for purified rAAV6.2-GFPand rAAV9-GFP vectors produced using TTT were 65.9% and 76.9%, respectively (data not shown) Analysis of the molar ratio of VP1:VP2:VP3 in the purified samples were consistent with those reported in the literature (Fig. 5d).These data show that rAAV vectors produced in our engineered CHO cells have good expression of capsid proteins with a high percentage of full AAV capsids.In a subsequent experiment, purified rAAV6.2-GFPand rAAV9-GFP vectors were examined for any residual infectious HSV-1 vectors in the PEG-Chloroform purified materials by inoculating 10 10 vg/mL of each vector on an HSV-1 complementing cell line (V27), with rHSV1-GFP vector (MOI 0.15 PFU/cell) as a positive control.No cytopathic effects appeared in wells inoculated with either purified rAAV6.2-GFPor rAAV9-GFP vectors up to 4 days post-infection.Conversely, the typical cytopathic effects, such as rounding of infected-cells and cell sheet detachment, appeared on day 2 post-infection in the well inoculated with rHSV1-GFP vector, with complete detachment of the cell sheet on day 3 post-infection (Supplementary Fig. S4).These data indicate that the inhouse developed PEG-Chloroform purification method is efficient in inactivating rHSV-1 vectors as indicated by no detection of residual infectious rHSV-1 in the purified rAAV, thereby ensuring that competent HSV-1 are not carried over into the manufacturing process.
Transduction is the process by which AAV infects cells.This process includes several steps: binding and penetration of cells, intracellular trafficking, uncoating, and nuclear transport.By utilizing in vitro transduction, we can understand the ability of the produced AAV to carry out the aforementioned steps.Our aim was to compare the in vitro potency of CHO-derived rAAVs to rAAVs produced using the standard TTT method.For infectivity, GFP expression from infected wells was recorded on day 5 post-infection using the IncuCyte.The rAAV6.2-GFP vectors produced in the CHO-HV-C1 clone (rAAV6.2-GFPCHO) and the rAAV6.2-GFPproduced in HEK293 cells (rAAV6.2-GFPTTT) showed comparable infectivity titers of 1.65 × 10 7 and 1.1 × 10 7 TCID 50 /mL, respectively (Fig. 6a).
To investigate the transduction efficiency of the two vector preparations mentioned above, Ad293 cells were infected with different MOI, ranging from 2 × 10 5 down to 1 × 10 3 vg/cell.The mean GFP expression was recorded on day 3 post-infection using the IncuCyte.The rAAV6.2-GFP-CHO and rAAV6.2-GFP-TTTshowed potent transduction at all of the MOI tested (Fig. 6b).These data indicate that rAAV6.2-GFP-CHOhas comparable in vitro infectivity and transduction activity to rAAV6.2-GFP-TTT.

Biodistribution of CHO-derived AAVs
The biodistribution of rAAV6.2-GFP-CHO, in parallel with the rAAV6.2-GFP-TTT,was assessed to determine if the in vivo behavior mimicked the in vitro data.Fifteen 3-week-old mice were divided into three groups (n = 5 per group).The mice were inoculated with either rAAV6.2-GFP-CHO(G1), rAAV6.2-GFP-TTT(G2), or PBS (G3).All mice were inoculated with either 10 11 vg rAAV or 100 µL of PBS intravenously in tail vein (TV), as per their grouping.A previous study showed that rAAV6 had high level of transduction in liver, skeletal muscle, kidney, lung, and heart in mouse post TV inoculation 30 .Therefore, these tissues were harvested from each inoculated animal three weeks post-inoculation.Samples were assessed for rAAV titer in homogenized tissue using qPCR (targeting the GFP gene), and histopathologic examination was performed using confocal microscopy.From the qPCR analysis, mice from G1 showed lower rAAV6.2-GFPgenome copy numbers than mice from G2 in all harvested tissue, without a significant statistical difference except for liver (p = 0.0012).(Fig. 7a).Thin liver tissue sections from the three groups were prepared and examined for GFP expression using confocal microscopy.Clear GFP signals were observed in the livers of all inoculated groups except G3 (Fig. 7b), as expected from the qPCR data.These data indicate that rAAV produced in engineered CHO cells show good biodistribution after tail vein injection.Additionally, the lower GFP in G1 vs. G2 liver sections may be due to the presence of impurities that cannot be eliminated with PEG-Chloroform method.

Reengineering CHO-HV-C1 cells for HSV-1 production
In order to replace adherent, complimenting V27 cells for large-scale rHSV-1 production, we aimed to engineer our CHO-HV-C1 cells to express ICP27, an immediate early protein necessary for expression of late HSV-1 gene products.We utilized two strategies to generate HSV-1-ICP27 producer clones in our engineered CHO cells.The first strategy was random integration using one plasmid (Fig. 8a).The second strategy was site-specific integration using CRISPR/Cas9 with two plasmids: the first plasmid had a cassette composed of CHO codonoptimized HSV-1 ICP27 ORF downstream of the CMV promoter and upstream of the SV40 polyA, with a puromycin selection cassette; the second plasmid encoded synthetic sgRNA for CHO exon 1 C12orf35 31 .The total length of the two cassettes was 4.1 kb flanked by the right and the left homology arms (750 bp each) (Fig. 8b).
Pools from either random or site-specific integration transfected cells were recovered after three weeks of double selection using 5 µg/mL of puromycin and 50 µM MSX.Twenty-four clones showing high growth, viability, and ICP27 expression were selected, including seven site-edited clones (clones 1-7) and 17 random-integrated clones (clones 8-24) (Fig. 8c).Random-integrated clone #11 (CHO-HV-ICP27-C11), which demonstrated the highest growth profile and ICP27 expression, was selected to be tested for rHSV-1 vector production.
The CHO-HV-ICP27-C11 clone was infected with rHSV-AAV9 (MOI = 10 PFU/cell) and incubated at 37 °C for 2 h for virus adsorption.After 2 h, infected cells were washed twice with sterile 1 × PBS to remove any viral residues.The infected cells were then incubated for 24 h at 37 °C in a 5% CO 2 static humidified incubator.On the second day, 1 mL of clarified supernatant from infected cells was passaged on to the V27 cells.The appearance of cell rounding and detachment of the infected cell sheet appeared two days post-infection.Additionally, expression of HSV-1 glycoprotein D (gD) was observed in the V27 cell lysate after infection with the rHSV-1 virus propagated in clone CHO-HV-ICP27-C11 (data not shown).These results indicates that CHO-HV-ICP27-C11 clone supports productive infection for rHSV-1 vectors.

Discussion
Gene therapy has great potential to address the lack of life-altering treatments or cures for many different diseases such as cystic fibrosis and heart failure.In recent years, gene therapy research has focused on the use of AAV vectors because of their long-lasting expression after delivery to target organs and their non-pathogenic profile in humans.Previous studies showed that rHSV-1-assisted rAAV production provides a highly efficient manufacturing method 3 .However, typical manufacturing of rAAV using the rHSV-1 system involves infection of virus production cells, such as BHK-21 or HEK293 cells, with two replication-deficient rHSV-1 vectors in a serum-supplemented medium.This approach is very expensive for large-scale production and may introduce adventitious viral agents and/or prions into the final product [46][47][48][49][50][51] .Another drawback of manufacturing rAAV using the rHSV-1 system is the challenge in generating sufficient rHSV-1 vector stock for clinical and commercial manufacturing, as the scale-up process currently depends on using adherent Vero cells expressing the HSV-1 ICP27 protein (V27 cells) 3 .CHO cells are the predominant mammalian cell type used to produce recombinant protein biologics, due to their ability to correctly fold, assemble, and modify recombinant proteins 22,32,33 .Certain animal cell types, such as swine testis (ST) and CHO cells, can bind HSV-1 virus efficiently but restrict viral entry 34,35 .In addition, it has been reported that ST and CHO cells become susceptible to HSV-1 entry upon expression of human cDNA encoding HVEM 24 .Here, we aimed to engineer suspension, serum-free-adapted CHO cells to produce different rAAVs using the HSV-1 helper system, a virus which naturally does not infect wildtype CHO cells.
We have successfully engineered suspension, serum-free-adapted CHO cells to express HVEM and/or Nectin-1 proteins, which are necessary for rHSV-1 entry and infection.All of the engineered stable CHO pools showed significant susceptibility to rHSV-1-GFP vector entry and infection, as evidenced by GFP expression, compared to the wild-type CHO cells.Interestingly, the CHO cell pools expressing HVEM only outperformed all other pools in GFP expression when infected with the rHSV1-GFP vector.These data agree with the previous publication, which reported that engineered CHO cells expressing the HVEM receptor became permissive to HSV-1 entry and infection 24 .In addition, it has been reported that HVEM receptor supports more HSV-1 infection in CHO cells than Nectin-1 35 .Interestingly, the HVEM-only pool also outperformed all other stable pools in producing high rAAV9-GFP vector titers from the cell culture medium 24 hpi, which encouraged us to generate clonal cells from this pool.
Following optimization of the infection parameters, clone CHO-HV-C1 produced various AAV serotypes at titers higher than previously reported in CHO cells (~ 10 10 vg/mL at 24 hpi).A MOI of 4:1 was optimum for production of rAAV6.2,rAAV8 and rAAV9 expressing GFP, with no additional improvement seen at higher MOIs for rAAV6.2(data not shown).Additionally, shifting temperature from 37 to 33 °C enhanced release of rAAVs into the medium, particularly for rAAV8 and rAAV9.These data are very promising, as the CHO-HSV-1 platform can produce high rAAV titers with a lower MOI than that required for BHK-21, where a MOI of 12:2 was reported as optimal 3 .The lower MOI for the CHO-HSV-1 platform will benefit the biomanufacturing capability, particularly for the yield and purification of HSV-1 from rAAVs.
It should also be noted that a sharp decrease in the CHO-HV-C1 cell viability post co-infection at 37 °C was observed during production of different AAV serotypes.However, this decrease was minimized by switching the infection temperature to 33 °C.It has been reported that the stability of rHSV-1 vectors was 2.5-fold greater at 33 °C than 37 °C, and rHSV-1 synchronous infection incubated at 33 °C produced twofold higher amounts of vectors than those incubated at 37°C 36 .Additionally, the Rep protein encoded in the rHSV-1 vector may exert a toxic effect on the infected CHO-HV-C1 cells after infection at 37 °C; however, this point needs further investigation.
Using the PEG-Chloroform purification method followed by concentration using the 100 kDa molecularweight cutoff (MWCO) sample concentrator, we were able to purify rAAVs produced in our engineered CHO cells in one day.The developed purification method is simple, cheap, and fast, with titers comparable with the more time-consuming iodixanol ultracentrifugation method, which agrees with other studies [27][28][29] .Moreover, no difference in either in vitro or in vivo transduction of rAAV purified with Chloroform/PEG or iodixanol ultracentrifugation was observed 37 .Additionally, rAAVs produced in our engineered CHO cells and purified with the PEG-Chloroform method showed potent in vitro transduction and infectivity comparable to those produced using the triple transient transfection 38 in HEK293 cells and purified with column chromatography 39 .
The AAV capsid gene produces viral proteins, VP1, VP2, and VP3, that assemble to form the T = 1 icosahedral capsid, consisting of 60 total VPs.The VP composition of the AAV capsid is estimated to be at a molar ratio of 1:1:10 7,40,41 , such that one AAV capsid likely has 5 VP1, 5 VP2, and 50 VP3.Interestingly, analysis of the www.nature.com/scientificreports/composition of the capsid protein ratio of rAAV6.2-GFPand rAAV9-GFP vectors produced in the CHO-HV-C1 cells using CE-SDS showed very comparable VP1:VP2:VP3 capsid ratios to those reported in the literature (data not shown).This result indicates that rAAVs produced in CHO-HV-C1 cells are functionally assembled, in contrast to the insect cell-baculovirus platform where genetic engineering and/or process optimization is required to enhance VP expression of some AAV serotypes.For example, early trials to adapt AAV5 for production using the insect cells-baculovirus platform showed low levels of VP1 incorporation into the capsid 52 that was improved later [53][54][55] .Examination of full and empty capsids for rAAVs produced in the CHO-HV-C1 clone showed higher full capsid percentage than what has been reported previously from an optimized rHSV-1 co-infection in BHK-21 cells 3 .However, the use of PEG in our protocol may have an impact on increasing the full capsid percentage, and future purification experiments using chromatography are necessary.We thought engineering CHO-HV-C1 cells to produce rHSV-1 stock would simplify the whole (AAV and rHSV-1) manufacturing process.Therefore, to address the current challenge in scaling-up rHSV-1 vector stock, we engineered CHO-HV-C1 cells to express the rHSV-1 ICP27 protein.Using an in-house medium, the top clone, CHO-HV1-ICP27-C11, showed productive infection for rHSV-1, as indicated by replication of CHOderived rHSV-1 on V27 cells.However, the production capacity of rHSV-1 vectors from CHO-HV1-ICP27-C11 was lower compared to V27 cells.Our preliminary expression profiles show that expression of some late HSV-1 viral proteins in the infected CHO-HV1-ICP27-C11 cells were lower than expression of early and intermediate viral genes.Additionally, CHO cells may not provide the elements necessary for the optimal expression of many HSV genes, particularly late genes, because CHO cells may express some inhibitory factors that interfere or block HSV-1 late viral genes expression.These findings agree with a previous study 24 .However, further investigation is necessary to understand if factors related to the in-house medium can potentially have some inhibitory effect for rHSV-1 vector production.

Concluding remarks
In summary, the engineered CHO-HV-C1 cells can produce multiple AAV serotypes at satisfactory titers using a serum-free medium with a lower MOI for HSV-1 vectors, compared to other studies that used a high MOI for HSV-1 vectors for AAV production in BHK-21 and HEK293 cells.We have observed that rHSV-1 MOI and incubation temperature after rHSV-1 co-infection are critical factors for production of a high rAAV titer in the CHO system.Moreover, low incubation temperature may have an impact on lowering the toxic effect of the rep gene on the producer cells.The optimum harvesting time for rAAV produced in the CHO cells was only 24 h post co-infection, which indicates the production and purification processes could be completed in as little as 2-3 days.The rAAVs produced in our engineered CHO cells showed high full capsid percentage and good capsid protein expression, transduction, and infectivity in vitro, with good biodistribution.There are some limitations in our study, including the need for optimization of purifying rAAV produced in CHO cells using chromatographic methods.Taken together, the HSV-based rAAV production platform in the engineered CHO cells provides a novel, scalable, serum-free manufacturing platform that will facilitate the production of future rAAV-based biotherapeutics in a low-cost manner.

Generation of stable CHO pools
The open reading frames (ORFs) of human HVEM and Nectin-1 were downloaded from the NCBI database (accessions U70321.1 and AF060231.1,respectively).Both HVEM and Nectin-1 ORFs were codon-optimized for expression in hamster cells using online tools (https:// www.idtdna.com/ Codon Opt [IDT] and https:// www.therm ofish er.com/ us/ en/ home/ life-scien ce/ cloni ng/ gene-synth esis/ genea rt-gene-synth esis/ geneo ptimi zer.html [Thermo Fisher Scientific, CA, USA], respectively).The HVEM was codon-optimized by IDT's tool using their property in-house algorithm with the default setting and human as the target species for the expression.The Nectin-1 was codon-optimized using GeneArt's tool using their default setting and human as the target species for expression as well.The delivered plasmids encoding synthetic HVEM or Nectin-1 were sub-cloned into an in-house plasmid downstream of the enhanced human cytomegalovirus (CMV) promoter or a synthetic promoter 42 , generating eight different constructs.All constructed plasmids encoded glutamine synthetase (GS) under the control of the SV40 promoter allowing for the selection of transfected cells in methionine sulfoximine (MSX).All final plasmids were verified by whole plasmid sequencing (Psomagen, MD, USA).Proprietary inhouse, suspension, serum-free-adapted CHO host cells were cultured in CD-CHO medium (10,743,029; Gibco, MD, USA), supplemented with 6 mM L-glutamine (part number 35050-06; Gibco, MD, USA), dextran sulphate (49,110; Sigma-Aldrich, MA, USA) and incubated in a 6% CO 2 humidified incubator at 37 °C and 120 rpm agitation.Viable cell density (VCD) and percent viability were measured daily using a Vi-Cell XR automated cell counter (Beckman Coulter, IN, USA).
Stably-transfected CHO pools were generated following an in-house protocol.In brief, eight aliquots of CHO cells (1 × 10 7 viable cells per aliquot) were pelleted for 5 min at 200 × g.Cell pellets were mixed with 7 µg of each purified linearized in-house plasmid and then transfected using the Amaxa cell line nucleofector Kit V (VCA-1003; Lonza, MA, USA), according to the manufacturer's instruction.After 24 h post-transfection, cell viability was measured, and MSX (M5379-1G; Sigma-Aldrich, MA, USA) was added to each pool for recombinant cell selection.

rHSV-1-GFP infection in stable CHO pools
The rHSV1-GFP was constructed by cloning the GFP flanked by AAV2 ITRs into HSV-1 DNA using standard molecular cloning techniques.All rHSV-1 vectors have been generated in V27 cells using homologous recombination.An aliquot (1 × 10 6 viable cells) from each recovered cell pool, as well as the CHO host cells, were infected with rHSV1-GFP (MOI = 10 PFU/cell) in a 6-well cell culture plate (3516; Corning, NY, USA) and incubated for 1 h at 37 °C in a humidified 5% CO 2 incubator.After one hour, infected cell pools were centrifuged for 5 min at 1200 rpm, and the virus supernatant was discarded.Infected cell pellets were resuspended into CD-CHO medium and incubated at 37 °C in a 5% CO 2 humidified incubator.Cells from each of the infected pools were imaged every 12 h for GFP expression using IncuCyte (Sartorius, Germany) with its default settings, for a total five time points.

rAAV9-GFP vector production in stable CHO pools
An aliquot of each cell pool (1 × 10

Generation of CHO-HVEM expressing clones and testing of rHSV1-GFP infection
Single-cell deposition was performed using a BD Influx cell sorter (BD Biosciences, CA, USA) according to Evans et al., 2015.In brief, an aliquot containing 3 × 10 6 viable CHO HVEM expressing cells were stained with 1:200 anti-human CD270 (HVEM)-PE antibody for 15 min in the dark at room temperature.Stained cells were washed twice with sterile PBS (RNBK5430; Sigma-Aldrich, MA, USA), pelleted at 200 × g for 5 min, and then resuspended in 1 mL sterile PBS.Single cells from the PE-gated fraction were deposited into each well of two 384-well plates (4588; Corning, NY, USA) containing in-house conditioned medium supplemented with MSX.The clonal cell lines were selected for testing for rHSV1-GFP infection, and the mean GFP expression was calculated from two time points (24 and 48 h post-infection) using IncuCyte as previously described.

Production of rAAV6.2-GFP vector in selected CHO-HVEM clones
The selected clones were further tested for rAAV6.2-GFPvector production using different MOIs for rHSV-AAV6.2rep/cap:rHSV-GFP.All infected clones were incubated at 37 °C in a 5% CO 2 humidified incubator with 120 rpm agitation for three days.An aliquot (1 mL) from each co-infected clone was harvested at 24 and 48 h post co-infection and centrifuged at 200 × g for 5 min.The harvested samples (cell pellets) were then prepared for rAAV titration using qPCR.In brief, infected cell pellets were collected, mixed with the AAV lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl), and subjected to three cycles of freeze/thawing in an isopropanol dry ice bath followed by centrifugation at 12,000 rpm for 30 min at 4 °C.After centrifugation, the supernatant was collected and the rAAV6.2-GFPtiters were determined using qPCR, as previously described.
The membrane was incubated with Supersignal West Pico Plus Substrate (34,580; ThermoFisher Scientific, MD, USA) before image detection using Amersham Imager 680 (GE Healthcare, UK).Purified rAAVs from the top CHO clone were visualized using mini-transmission electron microscopy (Mini-TEM; Vironova, Sweden).In brief, PEG-Chloroform purified rAAV6.2-GFPand rAAV9-GFP samples were placed on a 400-mesh glow-discharged carbon grid by first inverting the grid and placing it on top of a 10 μL droplet of rAAV, deposited on parafilm, for 30 s. Excess sample was blotted off by gently touching the edge of the grid against a Whatman filter paper.The grid was then washed twice with two 20 μL droplets of double distilled water.The sample-containing grid was then stained with a 20 μL droplet of 1.5% uranyl acetate for 10 s.Excess stain was blotted off by gently touching the edge of the grid against a Whatman filter paper.The rAAV samples were then visualized using a Mini-TEM instrument.

Detection of residual infectious rHSV-1 from purified CHO-derived rAAVs
V27 cells expressing a stable copy of the rHSV-1 ICP27 protein were seeded at 0.5 × 10 6 viable cells/well in a 6-well culture plate containing DMEM (11,995-065; Gibco, MD, USA) supplemented with 10% FBS and 500 µg/mL geneticin (10,131-035; Gibco, MD, USA) overnight.After 16 h, the cells were washed twice with sterile PBS and then infected with purified rAAV6.2-GFPvectors at a 1:100 dilution (~ 10 10 vg/mL).The rHSV1-GFP vector was used as positive control at MOI = 0.15 PFU/cell.Infected cells were incubated for 2 h at 37 °C for virus adsorption.After incubation, the excess virus was removed, and the infection medium (DMEM supplemented with 2% FBS) was added.The plate was incubated and monitored with IncuCyte for 4 days to capture any cytopathic effects.

In vitro infectivity of CHO-derived rAAVs
For infectivity analysis, Ad293 cells (240,085; Agilent, CA, USA) were seeded in a 96-well culture plate (2 × 10 4 cells/well) and incubated at 37 °C in a humidified, 5% CO 2 incubator overnight.Ten-fold dilutions of rAAV6.2-GFPproduced in either engineered CHO cells or HEK293 cells expressing adenovirus E1a and E1b proteins.rAAV6.2-GFPproduced in HEK293 cells using triple transfection.were prepared in 1X DMEM.Each virus dilution was used to infect four wells, and infected cells were incubated at 37 °C, 5% CO 2 for 5 days.On day 5 post-infection, infected cells were imaged for GFP expression using IncuCyte (default settings), and virus titers were calculated 44 and expressed as tissue culture infective dose 50 (TCID 50 ).For transduction analysis, Ad293 cells were cultured as mentioned above, and then infected with different MOI from CHO-derived rAAVs, or similar vectors produced by in HEK293 cells using the triple transient transfection method, as described above.

Biodistribution of CHO-derived rAAVs
All animal experiments were approved by the Institutional Animal Care and Use Committee of AstraZeneca (Gaithersburg, MD, USA).All animal experiments were performed in accordance with relevant guidelines and regulations.In addition, our study is reported in accordance with the ARRIVE guidelines.
Eight-week-old male C57bl/6 mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA).Mice were divided into three groups (n = 5 per group), and all mice were inoculated with 1 × 10 11 vg/100 µL of the appropriate rAAV vector (or saline) via the tail vein (TV) using an insulin syringe (Becton Dickinson, DC, USA).The inoculated mice were monitored daily for any clinical symptoms of illness.Three weeks post-injection, mice were euthanized via CO 2 inhalation and organs were harvested.Half of the harvested tissues were frozen in dry ice in a microcentrifuge tube for qPCR analysis, and the other half were fixed in 10% neutral buffered formalin for histology work.DNA extraction from the harvested tissues was performed using the All-Prep DNA/RNA Mini Kit (80,004; Qiagen, MD, USA), according to the manufacturer's instructions.The qPCR reactions were carried out on the QuantStudio 7 Flex (ThermoFisher Scientific, MA, USA) using in-house linearized plasmids, pAAV-GFP.The extracted genomic DNA (100 ng) was used as a template, with either specific GFP primers and probes (forward 5′-GAA CCG CAT CGA GCT GAA -3′, Reverse 5′-TGC TTG TCG GCC ATG ATA TAG-3′, and probe 5′/56-FAM/ATC GAC TTC/ZEN/AAG GAG GAC GGC AAC/3IABkFQ 3′.Cycling conditions were initial denaturation at 95 °C for 30 s, 40 cycles of 95 °C for 10 s, and annealing/extension at 60 °C for 20 s.For histology, thin liver tissue sections from the collected tissues were prepared using a Leica 3050 s microtome (Leica, Germany), stained, mounted, and assessed for GFP expression using confocal microscopy.

Engineering the CHO rHSV-1 vector producer cell line
Two strategies were used: (1) random integration, where HSV-1 ICP27 ORF (accession: AB235845.1)sequence was codon-optimized for hamster cells expression by IDT, chemically-synthesized, and then sub-cloned downstream the CMV promoter into an in-house plasmid encoding the puromycin cassette for selection; and (2) site-specific integration using CRISPR/Cas9 technology in which the exon1 from the C12Orf35 locus from CHO genome (accession: XM_027430029) was selected as one of the transcription hotspots 31 .The CRISPy bioinformatic tool 45 with the default parameters was used to select the sgRNA target sequence.The selected sgRNA

Figure 3 .
Figure 3. Testing CHO-HVEM expressing clones for HVEM surface expression and rHSV-1-GFP infection.(a) Mean Fluorescence Intensity of the top 24 CHO-HVEM expressing clones.(b)Mean GFP expression from the final 24 clones post rHSV-1 GFP infection using the IncuCyte.Data from the mean GFP expression were analyzed using the Kruskal-Wallis test with Dunn's multiple comparisons.Data (n = 2 per sample) presented as mean ± SD.

Figure 6 .
Figure 6.Infectivity of rAAVs produced in CHO-HV-C1 cells compared to HEK293.(a) Infectivity of purified rAAV6.2-GFPvector produced in either CHO-HV-C1 clone using rHSV-1 co-infection and/or HEK293 cells using triple transfection via TCID 50 .Data (n = 2 per sample) were analyzed with two-tailed Mann-Whitney test and expressed as mean ± SD.Individual biological replicates are shown for each group in the bar graph.(b) In vitro transduction of Ad293 cells with rAAV6.2-GFPproduced in either CHO-HV-C1 cells using rHSV-1 co-infection or HEK293 cells using triple transfection.Mock infection of cells used PBS alone.

Figure 7 .
Figure 7. Biodistribution of rAAVs-derived from CHO cells in mouse tissues.(a) Genome copy numbers of rAAV6.2GFP derived from CHO-HV-C1 or HEK293 isolated from heart, liver, and lung harvested tissues evaluated using qPCR.**p= 0.012.(b) Detection of GFP expression from harvested liver sections (G1, G2 and G3) using confocal microscopy.PBS was used as a negative control (mock).